Role of the Glutamyl α-Carboxylate of the Substrate Glutathione in the Catalytic Mechanism of Human Glutathione Transferase A1-1†

Biochemistry ◽  
2001 ◽  
Vol 40 (51) ◽  
pp. 15835-15845 ◽  
Author(s):  
Ann Gustafsson ◽  
Pär L. Pettersson ◽  
Leif Grehn ◽  
Per Jemth ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Multifunctional Role of Tyr 108 in the Catalytic Mechanism of Human Glutathione Transferase P1-1. Crystallographic and Kinetic Studies on the Y108F Mutant Enzyme†,‡

Biochemistry ◽  
1997 ◽  
Vol 36 (20) ◽  
pp. 6207-6217 ◽  
Author(s):  
Mario Lo Bello ◽  
Aaron J. Oakley ◽  
Andrea Battistoni ◽  
Anna P. Mazzetti ◽  
Marzia Nuccetelli ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Kinetic Studies ◽  
Mutant Enzyme

scholarly journals Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues

1996 ◽  
Vol 319 (1) ◽  
pp. 315-321 ◽  
Author(s):  
Kian-Leong TAN ◽  
Gareth CHELVANAYAGAM ◽  
Michael W. PARKER ◽  
Philip G. BOARD
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Cumene Hydroperoxide ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Serine Residue ◽  
Subsequent Removal ◽  
Carbonium Ion

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247–250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with 1-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of 1-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

Role of Lys-226 in the Catalytic Mechanism ofBacillus StearothermophilusSerine HydroxymethyltransferaseCrystal Structure and Kinetic Studies†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (18) ◽  
pp. 6929-6937 ◽  
Author(s):  
Siddegowda Bhavani ◽  
V. Trivedi ◽  
V. R. Jala ◽  
H. S. Subramanya ◽  
Purnima Kaul ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Kinetic Studies

scholarly journals The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

2021 ◽  
Vol 120 (3) ◽  
pp. 979-991
Author(s):  
Rebekah B. Stuart ◽  
Suzanne Zwaanswijk ◽  
Neil D. MacKintosh ◽  
Boontarikaan Witikornkul ◽  
Peter M. Brophy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Liver Fluke ◽  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Affinity Purification ◽  
Economic Loss ◽  
Differential Abundance ◽  
Parasitic Helminths

AbstractFasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

Study of the catalytic mechanism of a non-heme Fe catalyst: The role of the spin state of the iron

2021 ◽  
Vol 764 ◽  
pp. 138282
Author(s):  
Aikaterini Gemenetzi ◽  
Panagiota Stathi ◽  
Yiannis Deligiannakis ◽  
Maria Louloudi
Keyword(s):  
Spin State ◽  
Catalytic Mechanism ◽  
Fe Catalyst

Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization

2010 ◽  
Vol 431 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Natalia Fedulova ◽  
Françoise Raffalli-Mathieu ◽  
Bengt Mannervik
Keyword(s):  
Double Bond ◽  
Glutathione Transferase ◽  
Biological Species ◽  
Glutathione Transferases ◽  
Special Role ◽  
Primary Role ◽  
The Novel ◽  
Protective Function ◽  
Isomerase Activity

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

2001 ◽  
Vol 359 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Valeria MENCHISE ◽  
Catherine CORBIER ◽  
Claude DIDIERJEAN ◽  
Michele SAVIANO ◽  
Ettore BENEDETTI ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Chlamydomonas Reinhardtii ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Careful Analysis ◽  
Wild Type ◽  
Thioredoxin H ◽  
Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

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